It is considered that various bone diseases occur because, for example, calcium content of bones is decreased by the bone metabolism and insufficient osteogenesis. Typical bone diseases are, for example, fracture, osteomalacia, osteopenia, osteoporosis, back pain and low back pain. In those bone diseases, osteoporosis has a pathology caused by the following reasons: The bone mass is decreased as the balance of the bone resorption and the bone formation is lost by aging and, accordingly, the hone resorption is relatively increased to reduce the bone mass. As a result, the bone strength is decreased by the change in the fine structure of the bones to easily cause the fracture. Particularly in females, the bone mass is rapidly decreased after menopause, oophorectomy, etc. Osteoporosis not only causes the fractures or sharp pain but it also maces the patients bedridden, particularly in cases of elderly people. Under these circumstances, an effective cure is demanded for improving the quality of life in an aging society. Because it is difficult to cure patients with osteoporosis after the onset, the following points are now fully recognized: It is important to prevent this disease and it is also indispensable to start increasing of the amount of the bones in juvenile period. In addition, it is essential that nutrients required for the formation of bones and foods accelerating the formation of them must be taken everyday. As foods for strengthening the bones, calcium, magnesium and vitamin D are mainly taken nowadays. Casein phosphopeptide or the like for accelerating the absorption of calcium through the intestinal tracts is also used.
As the remedies for bone diseases such as osteoporosis, active vitamin D3, a female sex hormone (estrogen), calcitonin and ipriflavones are clinically used. Recently, a medicine for osteoporosis having an effect of polyisoprenoid derivatives typified by vitamin K2 for inhibiting the formation of osteoclasts was developed (Japanese Patent Kokai No. Hei 7-215849). There have been known a bone reinforcing agent containing casein phosphopeptide and genistein as the active ingredients (Japanese Patent Kokai No. 2001-302539); a composition for accelerating the bone formation, which is effective against osteoporosis and which contains saponin, daidzin, daidzein, genistin and genistein as the main active ingredients (Japanese Patent Kokai No 2000-191526); a composition for increasing the bone mass, which is effective against osteoporosis and which contains a Japanese horseradish extract as the active ingredient (Japanese Patent Kokai No Hei 10-279492); a remedy for bone diseases containing zinc acexamate as the active ingredient (Japanese Patent Kokai No. 10-218767); a composition for accelerating the bone formation and preventing the reduction in bone mineral density, which contains isoflavon as the main active ingredient (Japanese Patent Kokai No. Hei 10-114653); and an anti-osteoporotic composition containing reinforced vitamin K, and zinc (Japanese Patent Kokai No. Hei 10-36256).
On the other hand, β-cryptoxanthin (molecular weight: 552) is known as a carotenoid soluble in ethanol. β-Cryptoxanthin is contained in citrus fruits, particularly in Satsuma oranges in an amount of 1 to 2 mg in each orange. β-cryptoxanthin has characteristic properties of provitamin A. In addition, in the recent investigation of anticancer substances, it was found that β-cryptoxanthin has an anticancer effect stronger than that of β-carotene which is a carotenoid contained in green and yellow vegetables such as carrots and therefore, β-cryptoxanthin is becoming the center of attention (Biol. Pharm. Bull 18, 2, 227, 1995). Because β-cryptoxanthin is thus an important anti-carcinogenic component, techniques are now being developed for producing citrus fruits of a high quality having a β-cryptoxanthin content equal to that of the Satsuma oranges, for isolating genes for synthesizing β-cryptoxanthin (Japanese Patent Kokai Nos. Hei 11-155577 and Hei 11-46770), and for isolating a large amount of β-cryptoxanthin from the citrus fruits for the purpose of developing citrus fruits having an increased β-cryptoxanthin content and processed citrus fruit foods.
Methods for separating β-cryptoxanthin from citrus fruits such as Satsuma oranges are well known (Report of the Agricultural Department of Okayama University 69, 17-25, 1987, Tokyo Medical College Bulletin 18, 1-7, 1992 and (Journal of Food Biochemistry 18, 273-283, 1995) Recently, the following methods were proposed: a method for producing β-cryptoxanthin of a high purity which comprises the steps of pressing orange juice, obtaining an extract containing β-cryptoxanthin from the resultant precipitate width a solvent, hydrolyzing the extract, introducing the hydrolyzate into the first column filled with silica powder having an average particle diameter of 10 to 80 μm together with a primary development solvent at a linear velocity of at least 2 cm/min to separate a fraction containing. β-cryptoxanthin, removing the solvent, and introducing the isolated product into the second column filled with octadecylsilane silica having an average particle diameter of 10 to 80 μm together with a secondary development solvent at a linear velocity of at least 2 cm/min to separate the fraction containing at least 95% by weight of β-cryptoxanthin (Japanese Patent Kokai No. 2000-136181); a process for producing pulps having a high carotenoid content, which comprises the steps of squeezing citrus fruits, filtering or sieving the obtained juice, centrifuging the obtained the juice, adding an enzyme to the obtained precipitate, freezing the resultant mixture, thawing the mixture and dehydrating the obtained product; and a method for producing pulp containing an increased amount of carotenoid, β-cryptoxanthin, etc. and powder thereof which comprises the steps of repeating a process of adding water to a pulp having a high carotenoid content and dehydrating the pulp, and then drying and pulverizing the pulp (Japanese Patent Kokai No. 2000-23637).
For rapidly retrieving analogs which might have the activity of the lead compound, there is known a method for producing chemically possible combinatorial products from a large data base of finger prints having 3D multiple stereostructure and screening the products, which comprises the steps of temporarily binding the radical with a bulky space keeping group, registering the 3D model of the radical in a combinatorial ghost data base, detecting an optional atom having characteristic physical properties of pharmacophore type for an accessible optional molecular structure in the ghost data base; calculating all the distances between atoms relating to the whole stereostructure of the molecule, for the pair of the pharmacophore detected in each molecular structure to prepare the distance distribution density; preparing a stereostructure finger print vector including all the distance distribution density of the pair of the pharmacophore; defining the gauge function for each finger print for explaining the relative importance of the characteristics of the pharmacophore; preparing the finger print of the lead compound, comparing the finger print with each finger print of a possible library in the gauge function for making the lead compound maximum, and retrieving the possible library molecule for obtaining a mark below a specified threshold by the gauge function (Japanese Patent Publication No. 2001-521943).
It was reported that eight therapeutic agents now approved in Japan for bone diseases typified by osteoporosis are bone resorption-inhibitors (inhibiting the solution of bones) and also that only statin, which is a mevalonic acid synthetic inhibitors has the osteogenesis promoting effect. However, this finding is only on the gene level and, in fact, the osteogenesis promoting effect of statin was only weak. The object of the present invention is to provide an osteogenesis promoter having a remarkable effect of positively promoting osteogenesis and thus preventing/treating bone diseases, a preventive/a remedy for bone diseases such as osteoporosis having both of an osteogenesis-promoting effect and a bone resorption-inhibiting effect, and a method of screening an active ingredient for preventing/treating bone diseases by using a compound having both of an osteogenesis-promoting effect and a bone resorption-inhibiting effect as a lead compound.
The inventors have found that β-cryptoxanthin, which is contained in a large amount in peel and sarcocarp of Satsuma orange, has an osteogenesis-promoting effect and effect of preventing/treating bone diseases. Namely, the inventors made experiments wherein diaphysis and metaphysis tissue of a femur were cultured in a culture medium containing β-cryptoxanthin, then calcium level in the bone tissue, the amount of the expressed bone calcification accelerating enzyme and bone DNA level which is the index of the number of the cells in the bone tissue were determined to confirm a significant increase in all the cases. In the experiments, the inventors have found that β-cryptoxanthin accelerates the synthesis of protein in the cancellous bone in the femur tissue (tissue of the metaphysis) and cortical bone (tissue of the diaphysis) to promote the osteogenesis. The inventors cultured a bone tissue in the presence of both parathyroid hormone (PTH), having an effect of dissolving the bone mineral (bone resorption) and a physiological role of causing pathology of osteoporosis due to aging, and β-cryptoxanthin to confirm that the reduction in amount of calcium in the tissue of the diaphysis and tissue of the metaphysis can be significantly controlled. When the inventors orally administered β-cryptoxanthin to rats, all of calcium level in the diaphysis and metaphysis tissue the amount of the expressed bone calcification accelerating enzyme and bone DNA level which is the index of the number of cells in the bone tissue were significantly increased. The inventors have thus confirmed that the oral administration of β-cryptoxanthin effectively increases the bone mass. From those facts, it was made evident that β-cryptoxanthin accelerates the osteogenesis and also inhibits the bone resorption to exhibit the effect of keeping and/or increasing the bone mineral density and also to function as an anti-osteoporotic factor. It was confirmed by experiences that the effects confirmed in such a tissue culture system are almost 100% effective also in peroral experiments. The present invention has been completed on the basis of these findings.